Targeted mutagenesis of loop residues in the receptor-binding domain of the Bacillus thuringiensis Cry4Ba toxin affects larvicidal activity

Loop residues in domain 11 of Bacillus thuringiensis Cry delta-endotoxins have been demonstrated to be involved in insecticidal specificity. In this study, selected residues in loops beta6-beta7 (S387SPS390), beta8-beta9 (S-410, N-411, T-413, T-415, E-417 and G(418)) and beta10-beta11 (D454YNS457) in domain 11 of the Cry4Ba mosquito-larvicidal protein were changed individually to alanine by PCR-based directed mutagenesis. All mutant toxins were expressed in Escherichia coli JM109 cells as 130-kDa protoxins at levels comparable to the wild type. Only E coli cells that express the P389A, S410A. E417A, Y455A or N456A mutants exhibited a loss in toxicity against Aedes aegypti mosquito larvae of approximately 30% when compared to the wild type. In addition, K coli cells expressing double mutants, S410A/E417A or Y455A/N456A, at wild-type levels revealed a significantly higher loss in larvicidal activity of approximately 70%. Similar to the wild-type protoxin, both double mutant toxins were structurally stable upon solubilisation and trypsin activation in carbonate buffer, pH 9.0. These results indicate that S-410 and E417 in the beta8-beta9 loop, and Y-455 and N-456 in the beta10-beta11 loop are involved in larvicidal activity of the Cry4Ba toxin. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.